This dataset contains raw microsatellite genotypes for 304 Mottled Duskywing (Erynnis martialis) individuals across 10 putative populations. These data were used to investigate genetic diversity and population structure of the species across the species’ known Canadian populations and neighbouring populations in the United States (New York and Michigan). We characterized individuals at 21 polymorphic loci. Mottled Duskywing populations had moderate levels of genetic diversity, and populations within approximately 10 km of each other were genetically similar. These data informed the reintroduction of this endangered species to locations where it had become extirpated. Data from an additional sampled population that was included in the study (30 individuals) are governed by Indigenous data sovereignty and are therefore not included in this public repository.

We sampled Mottled Duskywing from 11 different populations between 2019-2022, with four of those populations sampled in two different years to assess temporal variation in allele frequencies and genetic drift. Tissue was obtained from either female adults used for captive rearing after they had expired, or non-lethal wing clip or leg samples collected in the field. Genomic DNA was extracted using a DNeasy Blood and Tissue Kit (Qiagen, Germantown, MD). Primers and multiplex PCR protocols described in Kroeze et al., 2022 were used to amplify microsatellites from all samples. Microsatellite loci were amplified using one fluorescently labelled primer per pair (6-FAM, VIC, NED, or PET label), and sized by fragment analysis on a 3730S capillary DNA analyzer, using LIZ-500 size standard (Applied Biosystems, Foster City, CA), at the University of British Columbia’s Sequencing and Bioinformatics Consortium (British Columbia, Canada). Genotypes were called using GeneMarker® HID software (SoftGenetics, State College, Pennsylvania, USA).